Overcoming the nutritional immunity by engineering iron-scavenging bacteria for cancer therapy.

Certain bacteria demonstrate the ability to target and colonize the tumor microenvironment, a characteristic that positions them as innovative carriers for delivering various therapeutic agents in cancer therapy. Nevertheless, our understanding of how bacteria adapt their physiological condition to the tumor microenvironment remains elusive. In this work, we employed liquid chromatography-tandem mass spectrometry to examine the proteome of E. coli colonized in murine tumors. Compared to E. coli cultivated in the rich medium, we found that E. coli colonized in tumors notably upregulated the processes related to ferric ions, including the enterobactin biosynthesis and iron homeostasis. This finding indicated that the tumor is an iron-deficient environment to E. coli. We also found that the colonization of E. coli in the tumor led to an increased expression of lipocalin 2 (LCN2), a host protein that can sequester the enterobactin. We therefore engineered E. coli in order to evade the nutritional immunity provided by LCN2. By introducing the IroA cluster, the E. coli synthesizes the glycosylated enterobactin, which creates steric hindrance to avoid the LCN2 sequestration. The IroA-E. coli showed enhanced resistance to LCN2 and significantly improved the anti-tumor activity in mice. Moreover, the mice cured by the IroA-E. coli treatment became resistant to the tumor re-challenge, indicating the establishment of immunological memory. Overall, our study underscores the crucial role of bacteria's ability to acquire ferric ions within the tumor microenvironment for effective cancer therapy.

Siderophore-mediated iron acquisition by Klebsiella pneumoniae.

Microbes synthesize and secrete siderophores, that bind and solubilize precipitated or otherwise unavailable iron in their microenvironments. Gram (-) bacterial TonB-dependent outer membrane receptors capture the resulting ferric siderophores to begin the uptake process. From their similarity to fepA, the structural gene for the Escherichia coli ferric enterobactin (FeEnt) receptor, we identified four homologous genes in the human and animal ESKAPE pathogen Klebsiella pneumoniae (strain Kp52.145). One locus encodes IroN (locus 0027 on plasmid pII), and three other loci encode other FepA orthologs/paralogs (chromosomal loci 1658, 2380, and 4984). Based on the crystal structure of E. coli FepA (1FEP), we modeled the tertiary structures of the K. pneumoniae FepA homologs and genetically engineered individual Cys substitutions in their predicted surface loops. We subjected bacteria expressing the Cys mutant proteins to modification with extrinsic fluorescein maleimide (FM) and used the resulting fluorescently labeled cells to spectroscopically monitor the binding and transport of catecholate ferric siderophores by the four different receptors. The FM-modified FepA homologs were nanosensors that defined the ferric catecholate uptake pathways in pathogenic strains of K. pneumoniae. In Kp52.145, loci 1658 and 4984 encoded receptors that primarily recognized and transported FeEnt; locus 0027 produced a receptor that principally bound and transported FeEnt and glucosylated FeEnt (FeGEnt); locus 2380 encoded a protein that bound ferric catecholate compounds but did not detectably transport them. The sensors also characterized the uptake of iron complexes, including FeGEnt, by the hypervirulent, hypermucoviscous K. pneumoniae strain hvKp1. IMPORTANCE: Both commensal and pathogenic bacteria produce small organic chelators, called siderophores, that avidly bind iron and increase its bioavailability. Klebsiella pneumoniae variably produces four siderophores that antagonize host iron sequestration: enterobactin, glucosylated enterobactin (also termed salmochelin), aerobactin, and yersiniabactin, which promote colonization of different host tissues. Abundant evidence links bacterial iron acquisition to virulence and infectious diseases. The data we report explain the recognition and transport of ferric catecholates and other siderophores, which are crucial to iron acquisition by K. pneumoniae.

In vitro and in silico studies of enterobactin-inspired Ciprofloxacin and Fosfomycin first generation conjugates on the antibiotic resistant E. coli OQ866153.

BACKGROUND: The emergence of antimicrobial resistance in bacterial pathogens is a growing concern worldwide due to its impact on the treatment of bacterial infections. The "Trojan Horse" strategy has been proposed as a potential solution to overcome drug resistance caused by permeability issues. OBJECTIVE: The objective of our research was to investigate the bactericidal activity and mechanism of action of the "Trojan Horse" strategy using enterobactin conjugated with Ciprofloxacin and Fosfomycin against the antibiotic-resistant Escherichia coli strain OQ866153. METHODOLOGY: Enterobactin, a mixed ligand of E. coli OQ866153, was conjugated with Ciprofloxacin and Fosfomycin individually to aid active absorption via specific enterobactin binding proteins (FepABCDG). The effectiveness of the conjugates was assessed by measuring their bactericidal activity against E. coli OQ866153, as well as their ability to inhibit DNA gyrase enzyme and biofilm formation. RESULTS: The Fe+3-enterobactin-Ciprofloxacin conjugate effectively inhibited the DNA gyrase enzyme (Docking score = -8.597 kcal/mol) and resulted in a lower concentration (25 µg/ml) required to eliminate supercoiled DNA plasmids compared to the parent drug (35 µg/ml; Docking score = -6.264 kcal/mol). The Fe+3-Enterobactin-Fosfomycin conjugate showed a higher inhibition percentage (100%) of biofilm formation compared to Fosfomycin (21.58%) at a concentration of 2 mg/ml, with docking scores of -5.481 and -3.756 kcal/mol against UDP-N acetylglucosamine 1-carboxyvinyltransferase MurA. CONCLUSION: The findings of this study suggest that the "Trojan Horse" strategy using enterobactin conjugated with Ciprofloxacin and Fosfomycin can effectively overcome permeability issues caused by efflux proteins and enhance the bactericidal activity of these drugs against antibiotic-resistant strains of E. coli.

Investigation of Siderophore-Platinum(IV) Conjugates Reveals Differing Antibacterial Activity and DNA Damage Depending on the Platinum Cargo.

The growing threat of bacterial infections coupled with the dwindling arsenal of effective antibiotics has heightened the urgency for innovative strategies to combat bacterial pathogens, particularly Gram-negative strains, which pose a significant challenge due to their outer membrane permeability barrier. In this study, we repurpose clinically approved anticancer agents as targeted antibacterials. We report two new siderophore-platinum(IV) conjugates, both of which consist of an oxaliplatin-based Pt(IV) prodrug (oxPt(IV)) conjugated to enterobactin (Ent), a triscatecholate siderophore employed by Enterobacteriaceae for iron acquisition. We demonstrate that l/d-Ent-oxPt(IV) (l/d-EOP) are selectively delivered into the Escherichia coli cytoplasm, achieving targeted antibacterial activity, causing filamentous morphology, and leading to enhanced Pt uptake by bacterial cells but reduced Pt uptake by human cells. d-EOP exhibits enhanced potency compared to oxaliplatin and l-EOP, primarily attributed to the intrinsic antibacterial activity of its non-native siderophore moiety. To further elucidate the antibacterial activity of Ent-Pt(IV) conjugates, we probed DNA damage caused by l/d-EOP and the previously reported cisplatin-based conjugates l/d-Ent-Pt(IV) (l/d-EP). A comparative analysis of these four conjugates reveals a correlation between antibacterial activity and the ability to induce DNA damage. This work expands the scope of Pt cargos targeted to the cytoplasm of Gram-negative bacteria via Ent conjugation, provides insight into the cellular consequences of Ent-Pt(IV) conjugates in E. coli, and furthers our understanding of the potential of Pt-based therapeutics for antibacterial applications.

Conjugation to Native and Nonnative Triscatecholate Siderophores Enhances Delivery and Antibacterial Activity of a ß-Lactam to Gram-Negative Bacterial Pathogens.

Developing new antibiotics and delivery strategies is of critical importance for treating infections caused by Gram-negative bacterial pathogens. Hijacking bacterial iron uptake machinery, such as that of the siderophore enterobactin (Ent), represents one promising approach toward these goals. Here, we report a novel Ent-inspired siderophore-antibiotic conjugate (SAC) employing an alternative siderophore moiety as the delivery vector and demonstrate the potency of our SACs harboring the ß-lactam antibiotic ampicillin (Amp) against multiple pathogenic Gram-negative bacterial strains. We establish the ability of N,N',N''-(nitrilotris(ethane-2,1-diyl))tris(2,3-dihydroxybenzamide) (TRENCAM, hereafter TC), a synthetic mimic of Ent, to facilitate drug delivery across the outer membrane (OM) of Gram-negative pathogens. Conjugation of Amp to a new monofunctionalized TC scaffold affords TC-Amp, which displays markedly enhanced antibacterial activity against the gastrointestinal pathogen Salmonella enterica serovar Typhimurium (STm) compared with unmodified Amp. Bacterial uptake, antibiotic susceptibility, and microscopy studies with STm show that the TC moiety facilitates TC-Amp uptake by the OM receptors FepA and IroN and that the Amp warhead inhibits penicillin-binding proteins. Moreover, TC-Amp achieves targeted activity, selectively killing STm in the presence of a commensal lactobacillus. Remarkably, we uncover that TC-Amp and its Ent-based predecessor Ent-Amp achieve enhanced antibacterial activity against diverse Gram-negative ESKAPE pathogens that express Ent uptake machinery, including strains that possess intrinsic ß-lactam resistance. TC-Amp and Ent-Amp exhibit potency comparable to that of the FDA-approved SAC cefiderocol against Gram-negative pathogens. These results demonstrate the effective application of native and appropriately designed nonnative siderophores as vectors for drug delivery across the OM of multiple Gram-negative bacterial pathogens.

"Off-Label Use" of the Siderophore Enterobactin Enables Targeted Imaging of Cancer with Radioactive Ti(IV).

The development of inert, biocompatible chelation methods is required to harness the emerging positron emitting radionuclide 45Ti for radiopharmaceutical applications. Herein, we evaluate the Ti(IV)-coordination chemistry of four catechol-based, hexacoordinate chelators using synthetic, structural, computational, and radiochemical approaches. The siderophore enterobactin (Ent) and its synthetic mimic TREN-CAM readily form mononuclear Ti(IV) species in aqueous solution at neutral pH. Radiolabeling studies reveal that Ent and TREN-CAM form mononuclear complexes with the short-lived, positron-emitting radionuclide 45Ti(IV), and do not transchelate to plasma proteins in vitro and exhibit rapid renal clearance in naïve mice. These features guide efforts to target the 45Ti isotope to prostate cancer tissue through the design, synthesis, and evaluation of Ent-DUPA, a small molecule conjugate composed of a prostate specific membrane antigen (PSMA) targeting peptide and a monofunctionalized Ent scaffold. The [45Ti][Ti(Ent-DUPA)]2- complex forms readily at room temperature. In a tumor xenograft model in mice, selective tumor tissue accumulation (8±5 %, n=5), and low off-target uptake in other organs is observed. Overall, this work demonstrates targeted imaging with 45Ti(IV), provides a foundation for advancing the application of 45Ti in nuclear medicine, and reveals that Ent can be repurposed as a 45Ti-complexing cargo for targeted nuclear imaging applications.

Exploring the Antibacterial Activity and Cellular Fates of Enterobactin-Drug Conjugates That Target Gram-Negative Bacterial Pathogens.

Siderophores are secondary metabolites utilized by bacteria to acquire iron (Fe), an essential transition metal nutrient. Fe levels in the host environment are tightly regulated and can be further restricted to starve invading bacterial pathogens in a host-defense process known as nutritional immunity. To survive and colonize the Fe-limited host environment, bacteria produce siderophores and express cognate siderophore transport machinery. These active transport pathways present an opportunity for selective and efficient drug delivery into bacterial cells, motivating decades of research on synthetic siderophore-antibiotic conjugates (SACs) as a Trojan-horse strategy for the development of targeted antibiotics.Enterobactin (Ent) is a triscatecholate siderophore produced and utilized by many Gram-negative bacteria, including all Escherichia coli and Salmonella species. Within these species, pathogenic strains cause a variety of human diseases including urinary tract infections, gastroenteritis, and sepsis. Infections caused by these Gram-negative pathogens can be difficult to treat because of the impermeability of the outer membrane (OM). This impermeability can be overcome by utilizing siderophores as drug delivery vectors for targeting Gram-negative pathogens. Ent is a promising delivery vector because it undergoes active transport across the OM mediated by the Ent uptake machinery after scavenging Fe(III) from the extracellular environment. Despite the well-elucidated chemistry and biology of Ent, its use for SAC development was hampered by the lack of an appropriate functional group for cargo attachment. Our laboratory addressed this need by designing and synthesizing monofunctionalized Ent scaffolds. Over the past decade, we have used these scaffolds to explore Ent-based SACs with a variety of drug warheads, including ß-lactam and fluoroquinolone antibiotics, and Pt(IV) prodrugs. Investigations of the antibacterial activities of these conjugates and their cellular fates have informed our design principles and revealed approaches to achieving enhanced antibacterial potency and pathogen-targeted activity. Collectively, our studies of Ent-drug conjugates have provided discoveries, understanding, and invaluable insights for future design and evaluation of SACs.In this Account, we present the story of our work on Ent-drug conjugates that began about ten years ago with the development of monofunctionalized Ent scaffolds and the design and synthesis of various conjugates based on these scaffolds. We describe the antibacterial activity profiles and uptake pathways of Ent-drug conjugates harboring traditional antibiotics and repurposed platinum anticancer agents as well as studies that address cellular targets and fates. Finally, we discuss other applications of monofunctionalized Ent scaffolds, including a siderophore-based immunization strategy. We intend for this Account to inspire further investigations into the fundamental understanding and translational applications of siderophores and siderophore-drug conjugates.

Functional and Evolutionary Integration of a Fungal Gene With a Bacterial Operon.

Siderophores are crucial for iron-scavenging in microorganisms. While many yeasts can uptake siderophores produced by other organisms, they are typically unable to synthesize siderophores themselves. In contrast, Wickerhamiella/Starmerella (W/S) clade yeasts gained the capacity to make the siderophore enterobactin following the remarkable horizontal acquisition of a bacterial operon enabling enterobactin synthesis. Yet, how these yeasts absorb the iron bound by enterobactin remains unresolved. Here, we demonstrate that Enb1 is the key enterobactin importer in the W/S-clade species Starmerella bombicola. Through phylogenomic analyses, we show that ENB1 is present in all W/S clade yeast species that retained the enterobactin biosynthetic genes. Conversely, it is absent in species that lost the ent genes, except for Starmerella stellata, making this species the only cheater in the W/S clade that can utilize enterobactin without producing it. Through phylogenetic analyses, we infer that ENB1 is a fungal gene that likely existed in the W/S clade prior to the acquisition of the ent genes and subsequently experienced multiple gene losses and duplications. Through phylogenetic topology tests, we show that ENB1 likely underwent horizontal gene transfer from an ancient W/S clade yeast to the order Saccharomycetales, which includes the model yeast Saccharomyces cerevisiae, followed by extensive secondary losses. Taken together, these results suggest that the fungal ENB1 and bacterial ent genes were cooperatively integrated into a functional unit within the W/S clade that enabled adaptation to iron-limited environments. This integrated fungal-bacterial circuit and its dynamic evolution determine the extant distribution of yeast enterobactin producers and cheaters.

A novel radiolabelled salmochelin derivative for bacteria-specific PET imaging: synthesis, radiolabelling and evaluation.

For specific imaging of bacterial infections we aimed at targeting the exclusive bacterial iron transport system via siderophore-based radiotracers. De novo synthesis and radiolabeling yielded the salmochelin-based PET radiotracer [68Ga]Ga-RMA693, which showed a favourable biodistribution and a bacteria-specific uptake in an animal model of Escherichia coli infection.

Yersiniabactin is a quorum-sensing autoinducer and siderophore in uropathogenic Escherichia coli.

Siderophores are secreted ferric ion chelators used to obtain iron in nutrient-limited environmental niches, including human hosts. While all Escherichia coli express the enterobactin (Ent) siderophore system, isolates from patients with urinary tract infections additionally express the genetically distinct yersiniabactin (Ybt) siderophore system. To determine whether the Ent and Ybt systems are functionally redundant for iron uptake, we compared the growth of different isogenic siderophore biosynthetic mutants in the presence of transferrin, a human iron-binding protein. We observed that Ybt expression does not compensate for deficient Ent expression following low-density inoculation. Using transcriptional and product analysis, we found this non-redundancy to be attributable to a density-dependent transcriptional stimulation cycle in which Ybt functions as an autoinducer. These results distinguish the Ybt system as a combined quorum-sensing and siderophore system. These functions may reflect Ybt as a public good within bacterial communities or as an adaptation to confined, subcellular compartments in infected hosts. This combined functionality may contribute to the extraintestinal pathogenic potential of E. coli and related Enterobacterales.IMPORTANCEPatients with urinary tract infections are often infected with Escherichia coli strains carrying adaptations that increase their pathogenic potential. One of these adaptations is the accumulation of multiple siderophore systems, which scavenge iron for nutritional use. While iron uptake is important for bacterial growth, the increased metabolic costs of siderophore production could diminish bacterial fitness during infections. In a siderophore-dependent growth condition, we show that the virulence-associated yersiniabactin siderophore system in uropathogenic E. coli is not redundant with the ubiquitous E. coli enterobactin system. This arises not from differences in iron-scavenging activity but because yersiniabactin is preferentially expressed during bacterial crowding, leaving bacteria dependent upon enterobactin for growth at low cell density. Notably, this regulatory mode arises because yersiniabactin stimulates its own expression, acting as an autoinducer in a previously unappreciated quorum-sensing system. This unexpected result connects quorum-sensing with pathogenic potential in E. coli and related Enterobacterales.

Uropathogenic Escherichia coli wield enterobactin-derived catabolites as siderophores.

Uropathogenic Escherichia coli (UPEC) secrete multiple siderophore types to scavenge extracellular iron(III) ions during clinical urinary tract infections, despite the metabolic costs of biosynthesis. Here, we find the siderophore enterobactin (Ent) and its related products to be prominent components of the iron-responsive extracellular metabolome of a model UPEC strain. Using defined Ent biosynthesis and import mutants, we identify lower molecular weight dimeric exometabolites as products of incomplete siderophore catabolism, rather than prematurely released biosynthetic intermediates. In E. coli, iron acquisition from iron(III)-Ent complexes requires intracellular esterases that hydrolyze the siderophore. Although UPEC are equipped to consume the products of completely hydrolyzed Ent, we find that Ent and its derivatives may be incompletely hydrolyzed to yield products with retained siderophore activity. These results are consistent with catabolic inefficiency as means to obtain more than one iron ion per siderophore molecule. This is compatible with an evolved UPEC strategy to maximize the nutritional returns from metabolic investments in siderophore biosynthesis.

Crystal structure and activity of a de novo enzyme, ferric enterobactin esterase Syn-F4.

Producing novel enzymes that are catalytically active in vitro and biologically functional in vivo is a key goal of synthetic biology. Previously, we reported Syn-F4, the first de novo protein that meets both criteria. Syn-F4 hydrolyzed the siderophore ferric enterobactin, and expression of Syn-F4 allowed an inviable strain of Escherichia coli (Δfes) to grow in iron-limited medium. Here, we describe the crystal structure of Syn-F4. Syn-F4 forms a dimeric 4-helix bundle. Each monomer comprises two long α-helices, and the loops of the Syn-F4 dimer are on the same end of the bundle (syn topology). Interestingly, there is a penetrated hole in the central region of the Syn-F4 structure. Extensive mutagenesis experiments in a previous study showed that five residues (Glu26, His74, Arg77, Lys78, and Arg85) were essential for enzymatic activity in vivo. All these residues are located around the hole in the central region of the Syn-F4 structure, suggesting a putative active site with a catalytic dyad (Glu26-His74). The complete inactivity of purified proteins with mutations at the five residues supports the putative active site and reaction mechanism. Molecular dynamics and docking simulations of the ferric enterobactin siderophore binding to the Syn-F4 structure demonstrate the dynamic property of the putative active site. The structure and active site of Syn-F4 are completely different from native enterobactin esterase enzymes, thereby demonstrating that proteins designed de novo can provide life-sustaining catalytic activities using structures and mechanisms dramatically different from those that arose in nature.

Iron acquisition by a commensal bacterium modifies host nutritional immunity during Salmonella infection.

During intestinal inflammation, host nutritional immunity starves microbes of essential micronutrients, such as iron. Pathogens scavenge iron using siderophores, including enterobactin; however, this strategy is counteracted by host protein lipocalin-2, which sequesters iron-laden enterobactin. Although this iron competition occurs in the presence of gut bacteria, the roles of commensals in nutritional immunity involving iron remain unexplored. Here, we report that the gut commensal Bacteroides thetaiotaomicron acquires iron and sustains its resilience in the inflamed gut by utilizing siderophores produced by other bacteria, including Salmonella, via a secreted siderophore-binding lipoprotein XusB. Notably, XusB-bound enterobactin is less accessible to host sequestration by lipocalin-2 but can be "re-acquired" by Salmonella, allowing the pathogen to evade nutritional immunity. Because the host and pathogen have been the focus of studies of nutritional immunity, this work adds commensal iron metabolism as a previously unrecognized mechanism modulating the host-pathogen interactions and nutritional immunity.

Affinity difference determines the assembly and interaction mode of anammox community reconstructed by siderophores.

Anammox community is the core of anammox process. The constancy of the anammox community determines the stability of the anammox process and the ability of withstand environmental impact. Community stability is influenced by the assembly and interaction mode of the community. This study aimed to explore the assembly, interaction mode, and stability of anammox community influenced by two siderophores (enterobactin and putrebactin) specific for Ca. Brocadia and Ca. Kuenenia as produced in our previous research. Siderophores improved the stability of the anammox community, among which vulnerability dropped by 30.02 % and 72.53 % respectively. Enterobactin and putrebactin altered the succession speed and assembly pattern of communities, with a respective increase of 9.77 % and 80.87 % in the deterministic process of anammox community assembly, respectively. Enterobactin and putrebactin reduced the dependence of Ca. Brocadia and Ca. Kuenenia on companion bacteria by 60 items and 27 items respectively. The affinity of different siderophore-Fe with bacterial membrane receptors caused variations in community reconstruction, with Ca. Brocadia and Ca. Kuenenia exhibiting the highest affinity with enterobactin-Fe (-11.4 kcal/mol) and putrebactin-Fe (-9.0 kcal/mol), respectively. This study demonstrated how siderophores can enhance the stability of anammox process by regulating assembly and interaction mode of anammox community, while also revealing the underlying molecular mechanisms.

Adding siderophores: A new strategy to reduce greenhouse gas emissions in composting.

Microbial community is the primary driver causing the greenhouse gas emissions in composting. Thus, regulating the microbial communities is a strategy to reduce them. Here, two different siderophores (enterobactin and putrebactin) were added, which could bind and translocate iron by specific microbes, to regulate the composting communities. The results showed that adding enterobactin enriched Acinetobacter and Bacillus with specific receptors by 6.84-fold and 6.78-fold. It promoted carbohydrate degradation and amino acid metabolism. This resulted in a 1.28-fold increase in humic acid content, as well as a 14.02% and 18.27% decrease in CO2 and CH4 emissions, respectively. Meanwhile, adding putrebactin boosted the microbial diversity by 1.21-fold and enhanced potential microbial interactions by 1.76-fold. The attenuated denitrification process led to a 1.51-fold increase in the total nitrogen content and a 27.47% reduction in N2O emissions. Overall, adding siderophores is an efficient strategy to reduce greenhouse gas emissions and promote the compost quality.

Evaluation of immunogenicity of enterobactin conjugate vaccine for the control of Escherichia coli mastitis in dairy cows.

Mastitis is the most common disease of dairy cows that incurs severe economic losses to the dairy industry. Currently, environmental mastitis pathogens are a major problem for most dairy farms. A current commercially available Escherichia coli vaccine does not prevent clinical mastitis and production losses, likely due to antibody accessibility and antigenic variation issues. Therefore, a novel vaccine that prevents clinical disease and production losses is critically needed. Recently a nutritional immunity approach, which restricts bacterial iron uptake by immunologically sequestering conserved iron-binding enterobactin (Ent), has been developed. The objective of this study was to evaluate the immunogenicity of the keyhole limpet hemocyanin-enterobactin (KLH-Ent) conjugate vaccine in dairy cows. Twelve pregnant Holstein dairy cows in their first through third lactations were randomized to the control or vaccine group, with 6 cows per group. The vaccine group received 3 subcutaneous vaccinations of KLH-Ent with adjuvants at drying off (D0), 20 (D21), and 40 (D42) days after drying off. The control group was injected with phosphate-buffered saline (pH 7.4) mixed with the same adjuvants at the same time points. Vaccination effects were assessed over the study period until the end of the first month of lactation. The KLH-Ent vaccine did not cause any systemic adverse reactions or reduction in milk production. Compared with the control group, the vaccine elicited significantly higher levels of serum Ent-specific IgG at calving (C0) and 30 d postcalving (C30), mainly its IgG2 fraction, which was significantly higher at D42, C0, C14, and C30 d, with no significant change in IgG1 levels. Milk Ent-specific IgG and IgG2 levels in the vaccine group were significantly higher on C30. Fecal microbial community structures were similar for both control and vaccine groups on the same day and shifted directionally along the sampling days. In conclusion, the KLH-Ent vaccine successfully triggered strong Ent-specific immune responses in dairy cows without significantly affecting the gut microbiota diversity and health. The results show that Ent conjugate vaccine is a promising nutritional immunity approach in control of E. coli mastitis in dairy cows.

Evaluation of immunogenicity and efficacy of the enterobactin conjugate vaccine in protecting chickens from colibacillosis.

Colibacillosis is one of the most common and economically devastating infectious diseases in poultry production worldwide. Innovative universal vaccines are urgently needed to protect chickens from the infections caused by genetically diverse avian pathogenic Escherichia coli (APEC). Enterobactin (Ent) is a highly conserved siderophore required for E. coli iron acquisition and pathogenesis. The Ent-specific antibodies induced by a novel Ent conjugate vaccine significantly inhibited the in vitro growth of diverse APEC strains. In this study, White Leghorn chickens were immunized with the Ent conjugate vaccine using a crossed design with two variables, vaccination (with or without) and APEC challenge (O1, O78, or PBS control), resulting in six study groups (9 to 10 birds/group). The chickens were subcutaneously injected with the vaccine (100 µg per bird) at 7 days of age, followed by booster immunization at 21 days of age. The chickens were intratracheally challenged with an APEC strain (108 CFU/bird) or PBS at 28 days of age. At 5 days post infection, all chickens were euthanized to examine lesions and APEC colonization of the major organs. Immunization of chickens with the Ent vaccine elicited a strong immune response with a 64-fold increase in the level of Ent-specific IgY in serum. The hypervirulent strain O78 caused extensive lesions in lung, air sac, heart, liver, and spleen with significantly reduced lesion scores observed in the vaccinated chickens. Interestingly, the vaccination did not significantly reduce APEC levels in the examined organs. The APEC O1 with low virulence only caused sporadic lesions in the organs in both vaccination and control groups. The Ent conjugate vaccine altered the bacterial community of the ileum and cecum. Taken together, the findings from this study showed the Ent conjugate vaccine could trigger a strong specific immune response and was promising to confer protection against APEC infection.

Synthesis of an Antimicrobial Enterobactin-Muraymycin Conjugate for Improved Activity Against Gram-Negative Bacteria.

Overcoming increasing antibiotic resistance requires the development of novel antibacterial agents that address new targets in bacterial cells. Naturally occurring nucleoside antibiotics (such as muraymycins) inhibit the bacterial membrane protein MraY, a clinically unexploited essential enzyme in peptidoglycan (cell wall) biosynthesis. Even though a range of synthetic muraymycin analogues has already been reported, they generally suffer from limited cellular uptake and a lack of activity against Gram-negative bacteria. We herein report an approach to overcome these hurdles: a synthetic muraymycin analogue has been conjugated to a siderophore, i. e. the enterobactin derivative EntKL , to increase the cellular uptake into Gram-negative bacteria. The resultant conjugate showed significantly improved antibacterial activity against an efflux-deficient E. coli strain, thus providing a proof-of-concept of this novel approach and a starting point for the future optimisation of such conjugates towards potent agents against Gram-negative pathogens.

Ligand-Promoted Surface Solubilization of TiO2 Nanoparticles by the Enterobactin Siderophore in Biological Medium.

Titanium dioxide nanoparticles (TiO2-NPs) are increasingly used in consumer products for their particular properties. Even though TiO2 is considered chemically stable and insoluble, studying their behavior in biological environments is of great importance to figure their potential dissolution and transformation. The interaction between TiO2-NPs with different sizes and crystallographic forms (anatase and rutile) and the strong chelating enterobactin (ent) siderophore was investigated to look at a possible dissolution. For the first time, direct evidence of anatase TiO2-NP surface dissolution or solubilization (i.e., the removal of Ti atoms located at the surface) in a biological medium by this siderophore was shown and the progressive formation of a hexacoordinated titanium-enterobactin (Ti-ent) complex observed. This complex was characterized by UV-visible and Fourier transform infrared (FTIR) spectroscopy (both supported by Density Functional Theory calculations) as well as electrospray ionization mass spectrometry (ESI-MS) and X-ray photoelectron spectroscopy (XPS). A maximum of ca. 6.3% of Ti surface atoms were found to be solubilized after 24 h of incubation, releasing Ti-ent complexes in the micromolar range that could then be taken up by bacteria in an iron-depleted medium. From a health and environmental point of view, the effects associated to the solubilization of the E171 TiO2 food additive in the presence of enterobactin and the entrance of the Ti-enterobactin complex in bacteria were questioned.

Siderophore Immunization Restricted Colonization of Adherent-Invasive Escherichia coli and Ameliorated Experimental Colitis.

Inflammatory bowel diseases (IBD) are characterized by chronic inflammation of the gastrointestinal tract and profound alterations to the gut microbiome. Adherent-invasive Escherichia coli (AIEC) is a mucosa-associated pathobiont that colonizes the gut of patients with Crohn's disease, a form of IBD. Because AIEC exacerbates gut inflammation, strategies to reduce the AIEC bloom during colitis are highly desirable. To thrive in the inflamed gut, Enterobacteriaceae acquire the essential metal nutrient iron by producing and releasing siderophores. Here, we implemented an immunization-based strategy to target the siderophores enterobactin and its glucosylated derivative salmochelin to reduce the AIEC bloom in the inflamed gut. Using chemical (dextran sulfate sodium) and genetic (Il10-/- mice) IBD mouse models, we showed that immunization with enterobactin conjugated to the mucosal adjuvant cholera toxin subunit B potently elicited mucosal and serum antibodies against these siderophores. Siderophore-immunized mice exhibited lower AIEC gut colonization, diminished AIEC association with the gut mucosa, and reduced colitis severity. Moreover, Peyer's patches and the colonic lamina propria harbored enterobactin-specific B cells that could be identified by flow cytometry. The beneficial effect of siderophore immunization was primarily B cell-dependent because immunized muMT-/- mice, which lack mature B lymphocytes, were not protected during AIEC infection. Collectively, our study identified siderophores as a potential therapeutic target to reduce AIEC colonization and its association with the gut mucosa, which ultimately may reduce colitis exacerbation. Moreover, this work provides the foundation for developing monoclonal antibodies against siderophores, which could provide a narrow-spectrum strategy to target the AIEC bloom in Crohn's disease patients. IMPORTANCE Adherent-invasive Escherichia coli (AIEC) is abnormally prevalent in patients with ileal Crohn's disease and exacerbates intestinal inflammation, but treatment strategies that selectively target AIEC are unavailable. Iron is an essential micronutrient for most living organisms, and bacterial pathogens have evolved sophisticated strategies to capture iron from the host environment. AIEC produces siderophores, small, secreted molecules with a high affinity for iron. Here, we showed that immunization to elicit antibodies against siderophores promoted a reduction of the AIEC bloom, interfered with AIEC association with the mucosa, and mitigated colitis in experimental mouse models. We also established a flow cytometry-based approach to visualize and isolate siderophore-specific B cells, a prerequisite for engineering monoclonal antibodies against these molecules. Together, this work could lead to a more selective and antibiotic-sparing strategy to target AIEC in Crohn's disease patients.